Supplementary Materials for

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1 advances.sciencemag.org/cgi/content/full/2/2/e /dc1 Supplementary Materials for Hidden in plain sight: Cryptic and endemic malaria parasites in North American white-tailed deer ( ) Ellen S. Martinsen, Nancy McInerney, Heidi Brightman, Ken Ferebee, Tim Walsh, William J. McShea, Tavis D. Forrester, Lisa Ware, Priscilla H. Joyner, Susan L. Perkins, Emily K. Latch, Michael J. Yabsley, Joseph J. Schall, Robert C. Fleischer The PDF file includes: Published 5 February 2016, Sci. Adv. 2, e (2016) DOI: /sciadv Table S1. Mosquito species screened by nested PCR for Plasmodium infection for the study. Table S2. County and state locations for the wild ungulate species sampled in our study and proportion found to be infected with malaria parasites at each site by nested PCR. Table S3. Collection animals screened at the NZP s campuses in Washington, DC, and Front Royal, VA, for malaria parasite infection by nested PCR. Table S4. GenBank accession numbers for Plasmodium sequences generated from WTD and A. punctipennis and used in the phylogenetic analyses in this study.

2 Supplementary Materials Supplementary Tables Table S1. Mosquito species screened by nested PCR for Plasmodium infection for the study. Sampling sites included the National Zoological Park in Washington, D.C. (DC), and sites within San Diego County, California (CA). Mammalian malaria parasites were only isolated from Anopheles punctipennis mosquitoes in this study. Mosquito species and sampling site Number of individuals screened by PCR Aedes albopictus (DC) 21 Aedes aegypti (DC) 4 Aedes japonicas japonicus (DC) 145 Aedes triseriatus (DC) 16 Aedes vexans (DC) 35 Anopheles barberi (DC) 1 Anopheles crucians (DC) 1 Anopheles punctipennis (DC) 35 Anopheles quadrimaculatus (DC) 3 Coquillettidia perturbans (DC) 1 Culex erythrothorax (CA) 299 Culex erraticus (DC) 6 Culex pipiens or Culex restuans (DC)* 1138 Culex quinquefasciatus (CA) 59 Culex salinarus (DC) 14 Culex stigmatosoma (CA) 11 Culex tarsalis (CA) 128 Culex territans (DC) 5 Culiseta incidens (CA) 37 Culiseta melanura (DC) 1 Culiseta morsitans (DC) 3 Culiseta particeps (CA) 1 Orthopodomyia signifera (DC) 1 Psorophora columbia (DC) 2 Psorophora ferox (DC) 1 Uranotaenia sapphirina (DC) 10 *Culex pipiens and Culex restuans were distinguished by PCR analysis for infected individuals.

3 Table S2. County and state locations for the wild ungulate species sampled in our study and proportion found to be infected with malaria parasites at each site by nested PCR. State abbreviations are as follows: AL=Alabama, AR=Arkansas, CO=Colorado, CT=Connecticut, DC=District of Columbia, GA=Georgia, ID=Idaho, KS=Kansas, KY=Kentucky, LA=Louisiana, MS=Mississippi, NC=North Carolina, NY=New York, SC=South Carolina, TX=Texas, VA= WV=West CA=California, MT=Montana, SD=South Dakota, AZ=Arizona, and OR=Oregon.

4 Host species County State #infected/ #sampled White-tailed deer Marshall AL 0/5 Shelby AL 2/5 Hemstead AR 0/5 Lawrence AR 0/6 Larimer CO 0/1 Fairfield CT 0/4 Washington DC 3/20 McIntosh GA 1/12 Latah ID 0/1 Crawford KS 0/1 Elk KS 0/1 Johnson KS 0/1 Labette KS 0/1 McPherson KS 0/1 Osage KS 0/1 Saline KS 0/1 Ballard KY 3/11 Boone KY 0/4 Hardin KY 1/5 Knott KY 0/2 Madison KY 1/5 Owen KY 0/4 Avoyelles Parish LA 1/5

5 Concordia Parish LA 0/5 Adams MS 0/5 Dare NC 2/11 Westchester County NY 4/76 Beaufort SC 1/9 Bosque TX 0/3 Coke TX 0/4 Duval TX 0/1 Henderson TX 0/1 Hopkins TX 0/1 Tarrant TX 0/1 Tom Greene TX 0/1 Warren VA 12/46 Hardy WV 2/5 Knott WV 0/2 Lewis WV 3/5 Mason WV 1/5 Mineral WV 0/5 Pocohontas WV 1/5 Taylor WV 1/5 Upshur WV 2/5 Witt WV 0/5 Elk Contra Costa CA 0/3 Kern CA 0/3 Solano CA 0/3

6 Gallatin MT 0/2 Custer SD 0/30 Pronghorn Coconino AZ 0/12 Maricopa AZ 0/12 Navajo AZ 0/12 Yavapai AZ 0/12 Mule/Black-tailed deer Cochese AZ 0/29 Pima AZ 0/29 Benton OR 0/2 Lincoln OR 0/1 Table S3. Collection animals screened at the NZP s campuses in Washington, DC, and Front Royal, VA, for malaria parasite infection by nested PCR. Collection species Number of Individuals Screened Alpaca (Vicugna pacos) 2 Dama Gazelle (Nanger dama) 4 Domestic Cow (Bos taurus) 2 Domestic Donkey (Equus africanus asinus) 4 Domestic Goat (Capra aegagrus hircus) 4 Eld s Deer (Panolia eldii) 6 Przewalski s Horse (Equus ferus przewalskii) 5 Scimitar-horned Oryx (Oryx dammah) 8 Tufted Deer (Elaphodus cephalophus) 4

7 Table S4. GenBank accession numbers for Plasmodium sequences generated from WTD and A. punctipennis and used in the phylogenetic analyses in this study. GenBank accession nos. Parasite lineage Host species Sampling location cytb co1 clpc asl (ANPUN01) Anopheles punctipennis Washington, D.C., KU KU KU KU (ANPUN02) Anopheles punctipennis Washington, D.C., KU KU KU KU (ODVIR01) KU KU KU KU (ODVIR02) KU KU KU KU (ODVIR03) KU N/A N/A N/A (ODVIR04) KU N/A N/A N/A (ODVIR05) KU N/A N/A N/A (ODVIR06) KU N/A N/A N/A